Method of thrombolysis by local delivery of reversibly inactivated acidified plasmin

ABSTRACT

Methods of thrombolysis that allow the use of a fibrinolytic composition comprising reversibly inactivated acidified plasmin and the localized delivery of the plasmin to a vascular thrombotic occlusion are disclosed. Further disclosed is a method for administering a therapeutic dose of a fibrinolytic composition substantially free of plasminogen activator to a human or animal having a vascular thrombotic occlusion. The fibrinolytic composition includes a reversibly inactivated acidified plasmin substantially free of plasminogen activator. Intravascular catheter delivery of the fibrinolytic composition directly into or in the immediate vicinity of the thrombus is disclosed to minimize the systemic degradation of fibrin while retaining the maximum plasmin activity against the thrombus.

REFERENCE TO RELATED APPLICATION

This application is a continuation of International ApplicationPCT/US00/31115 published in English on Nov. 13, 2000, which in turn is acontinuation-in-part of U.S. patent application Ser. No. 09/438,331,filed Nov. 13, 1999 now U.S. Pat. No. 6,355,243.

FIELD OF THE INVENTION

The present invention relates generally to a method of thrombolysisusing reversibly inactivated acidified plasmin that is substantiallyfree of plasminogen activators and its local delivery of the acidifiedplasmin proximal to, or directly within, a thrombus. The therapeuticmethod is particularly applicable to the dissolution of thrombi wherevercatheter-directed delivery is feasible.

BACKGROUND

Thrombotic disease is a major cause of morbidity and mortality in themodern world. Acute myocardial infarction and ischemic stroke are thefirst and third causes of death and disability in Western societies.Occlusive thrombosis results in loss of blood flow to vital organsproducing local oxygen deprivation, cell necrosis and loss of organfunction. There are major benefits to the rapid destruction of athrombus, resulting in early re-canalization: it prevents cell death,reduces infarct size, preserves organ function, and reduces early andlate mortality. Thrombolytic therapy is now administered to more than750,000 patients per year worldwide, while many times that number couldpotentially benefit from such treatment.

Thrombolytic agents now used in the lysis of occlusive blood clots areplasminogen activators. Several different plasminogen activators arecurrently available for immediate clinical use and several newgeneration plasminogen activators are the subject of clinical testing:tissue plasminogen activator, tPA, and its second generation successorTNK-tPA, RETEPLASE™ (a deletion mutant of tPA), single chainurokinase-type plasminogen activator (scuPA, or pro-urokinase),urokinase (UK), streptokinase (SK), and anisoylated plasminogenstreptokinase activator complex (APSAC). tPA, scuPA, and UK are normallyto be found at low levels in humans. Streptokinase is a bacterial enzymewith a powerful thrombolytic activity. APSAC is an anisolatedstreptokinase-plasminogen complex. In all cases the plasminogenactivators are capable of converting the zymogen plasminogen to theactive protease plasmin. The advantage offered by tPA and scuPA (and, toa lesser degree, APSAC) is that their activation of plasminogen isfibrin specific; binding to fibrin is a prerequisite for their fullproteolytic activity to be realized (Haber et al., 1989). Urokinase andstreptokinase can activate plasminogen in the absence of fibrin. Suchvariation in the affinity for fibrin has important consequences as tothe extent to which systemic bleeding occurs in animal models; thesedifferences, however, have not been appreciated clinically.

Plasminogen activators universally exert their thrombolytic potential byactivating circulating zymogen plasminogen into plasmin. Plasmin, theprinciple fibrinolytic enzyme in mammals is a serine protease withtrypsin-like specificity that is derived from the inactive zymogenprecursor plasminogen circulating in plasma. Plasminogen itself is a 791amino acid polypeptide having an N-terminus glutamate residue.Plasminogen activators such as tissue plasminogen activator (tPA) orurokinase will cleave the single-chain plasminogen molecule, to produceactive plasmin, at the Arg⁵⁶¹-Val⁵⁶² peptide bond. The resulting twopolypeptide chains of plasmin are held together by two interchaindisulfide bridges. The light chain of 25 kDa carries the catalyticcenter and is homologous to trypsin and other serine proteases. Theheavy chain (60 kDa) consists of five triple-loop kringle structureswith highly similar amino acid sequences. Some of these kringles containso-called lysine-binding sites that are responsible for plasminogen andplasmin interaction with fibrin, α₂-antiplasmin or other proteins.

The inherent problem with the therapeutic use of existing plasminogenactivators such as tPA, UK and SK is bleeding complications associatedwith their use, including, for example, gastrointenstinal hemorrhage inup to 20% of patients. Intracranial hemorrhage, which is clinically themost serious, is a frequent and lethal side effect of currentthrombolytic therapy, and occurs in approximately 1% of patients. Themechanism for bleeding is multifactorial and is believed to be due tounmasking of a vascular injury by lysis of a protective hemostatic plugand consequent loss of vascular integrity. This is combined with thesystemic activation of the fibrinolytic system and its attendantdepletion of clotting factors. The focus of much recent research hasbeen on generating modified plasminogen activators that exhibit improvedfibrin specificity; this was expected to reduce the amount of bleedingcomplications. In some cases, these novel activators tend to preservethe circulating levels of such clotting factors such as fibrinogen,Factors VIII and V, plasminogen, and α₂-antiplasmin. They specificallytarget and bind to the fibrin molecules that reside in a thrombus, andwill only act upon plasminogen when so bound. This has the result thatplasminogen is cleaved to the active protease plasmin only in thevicinity of the thrombosis and the level of non-specific systemiccleavage of fibrin is reduced. However, the number of bleedingcomplications with these new plasminogen activators remains significant.

The clinical success for thrombolytic drugs such as tissue plasminogenactivator (tPA), streptokinase and urokinase in reducing the extent of athrombotic occlusion of a vascular vessel is established. Plasminogenactivators have therefore become a treatment of choice in the managementof acute myocardial infarction and some other thrombotic conditions.Nevertheless, various disorders, including myocardial infarction,occlusive stroke, deep venous thrombosis and peripheral arterialdisease, remain a serious clinical problem and the known plasminogenactivators currently used suffer from several limitations that impacttheir overall usefulness in the elimination of a thrombus. In myocardialinfarction, vascular flow is restored within 90 minutes in approximately50% of patients, while acute coronary re-occlusion occurs in roughly 10%of patients. Coronary recanalization requires on average 45 minutes ormore. Residual mortality, principally due to intracerebral hemorrhage,is still at least 50% of the mortality level in the absence ofthrombolysis treatment.

Most research in the area of thrombolytics has focused on improving theefficacy and fibrin specificity of existing plasminogen activators aswell as finding new ones. Much of this effort has concentrated ontargeting the plasminogen activators to the fibrin that forms thescaffold of a thrombus and to improve the pharmacokinetics of theactivators when administered into the blood stream. This would allowtheir administration as bolus doses rather than as a continuous deliverythat prolongs exposure of the patient to the active agent, and theaccompanying risk of undesirable systemic hemorrhage.

Based on the results of Phase II clinical trials with targetedplasminogen activators such as TNK-tPA, vampire bat salivary plasminogenactivator, however, the anticipated improvement in safety profiles ofthe new plasminogen activators have not been realized clinicallyfollowing thrombolytic therapy. The percentage of moderate and majorbleeding episodes, including intracranial hemorrhage and stroke, werecomparable with the original unmodified tPA. The clogged arteries werenot opened earlier, and the rate of re-occlusions remained unchanged. Itappeared that the only benefit these activators have is the prolongedplasma half-life and the possibility of bolus administration.

Another problem with plasminogen activators is that they have limitedefficacy in the treatment of long clots found in peripheral arterialocclusions (PAO). These thrombi are typically aged and can grow to asignificant size. The average size of peripheral thrombi found both inthe native arteries and grafts is 31±11 cm. Aged and retracted thrombiare deficient in plasminogen, which therefore limits the susceptibilityof old thrombi to plasminogen activator-induced thrombolysis. It isquite common for a patient with a PAO to be treated for 24 hours or morewith urokinase and even after this prolonged period not to have completepatency of the vessel. The problem is greater with the delivery of theexisting thrombolytic agents via catheter directly into the interior ofthe thrombus where there are reduced levels of the plasminogensubstrate.

A fundamentally different approach to avoid the problems associated withthe systemic administration of a plasminogen activator to generatesufficient plasmin at the site of the thrombus, is to administer plasminitself directly to the patient. This is because plasmin is ultimatelythe enzyme mediating thrombolysis initiated by plasminogen activators.Direct delivery of active plasmin directly into retracted thrombi wouldcircumvent the inherent plasminogen deficiency of these thrombi andprovide predictable, rapid and effective thrombolysis irrespective ofplasminogen content.

Reich et al. in U.S. Pat. No. 5,288,489 discloses a fibrinolytictreatment that includes parenteral administration of plasmin into thebody of a patient. The concentration and time of treatment weresufficient to allow active plasmin to attain a concentration at the siteof an intravascular thrombus that is sufficient to lyse the thrombus orto reduce circulating fibrinogen levels. Reich et al., however, requiregeneration of the plasmin from plasminogen immediately prior to itsintroduction into the body.

In contrast, Jenson in U.S. Pat. No. 3,950,513 discloses a porcineplasmin preparation that is asserted to be stabilized at low pH. Theplasmin solution is neutralized before systemic administration to humansfor thrombolytic therapy.

Yago et al. in U.S. Pat. No. 5,879,923 discloses plasmin compositionsuseful as a diagnostic reagent. The compositions of Yago et al. consistof low concentrations of plasmin at a neutral pH and an additionalcomponent that may be 1) an oligopeptide comprising at least two aminoacids, or 2) at least two amino acids, or 3) a single amino acid and apolyhydric alcohol.

Plasmin represents a second mechanistic class of thrombolytic agents,distinct from the class of plasminogen activators. Although plasmin hadbeen investigated as a potential thrombolytic agent, numerous technicaldifficulties have prevented effective clinical use of this fibrinolyticenzyme. These difficulties included the challenge of preparing pureplasmin that is free of all functional traces of the plasminogenactivator used to generate plasmin from the inactive precursor,plasminogen. The thrombolytic activity of these earlier plasminpreparations was eventually attributed to the presence of contaminatingplasminogen activators rather than to plasmin itself. The contaminatingplasminogen activators, however, also triggered systemic bleeding atsites other than at the targeted thrombus. An additional drawback ofstreptokinase used for plasmin preparations is that its presence in apreparation of plasmin often causes adverse immune responses includingfever and anaphylactic shock.

The most important limitation to the clinical use of plasmin is that, asa serine protease with broad specificity, it is highly prone toautodegradation and loss of activity at physiological pH. This providessevere challenges to the production of high-quality stable plasminformulation suitable for prolonged periods of storage prior to use, andto safe and effective administration of plasmin to human patientssuffering from occlusive thrombi.

What is needed, therefore, is a method of administering a stable form ofactive plasmin that is substantially free of plasminogen activators andwhich is active upon encountering a targeted vascular thromboticocclusion.

What is also needed is a method of lysis of thrombi that directlydelivers an activatable form of active plasmin via a catheter into theinterior of the thrombus.

What is further needed is a method of thrombolysis in which anadministered thrombolytic agent is restricted to the thrombotic site andwhich exhibits reduced systemic, and especially intracranial,hemorrhage.

These and other objectives and advantages of the invention will becomefully apparent from the description and claims that follow or may belearned by the practice of the invention.

SUMMARY OF THE INVENTION

This invention relates to the discovery that thrombolytic therapy can beimproved by the administration of reversibly inactivated acidifiedplasmin directly into, or proximal to, a thrombus. Purified plasmin isunstable at physiological pH due to autodegradation and, therefore,plasmin has not been readily available for therapeutic administration.The present invention provides a method of administering a therapeuticdose of a fibrinolytic composition to a human or animal having avascular thrombotic occlusion, comprising administering a therapeuticdose of a pharmaceutically acceptable reversibly inactivated acidifiedfibrinolytic composition substantially free of plasminogen activator.The present invention further provides a method of administering atherapeutic dose of an reversibly inactivated acidified fibrinolyticcomposition comprising plasmin and a pharmaceutically acceptableacidified carrier, wherein the pharmaceutically acceptable carrier has alow buffering capacity.

The present invention also provides a method of administering atherapeutic dose of a reversibly inactivated acidified fibrinolyticcomposition, wherein the fibrinolytic composition comprises plasmin anda pharmaceutically acceptable acidified carrier, wherein thepharmaceutically acceptable acidified carrier comprises water and apharmaceutically acceptable acid selected from the acid forms ofcarboxylic acids, amino acids or other compounds having a low bufferingcapacity and the acidified carrier has a pH less than about 4.0.

The present invention provides a method of administering a therapeuticdose of a reversibly inactivated acidified plasmin and apharmaceutically acceptable acidified carrier, further comprising apharmaceutically acceptable stabilizer such as, but not limited to, asugar or a polyhydric alcohol.

In one aspect of the present invention, the fibrinolytic compositioncomprises a reversibly inactivated acidified serine proteasesubstantially free of its respective activator, a low buffering capacitybuffer, and optionally, a stabilizing agent. Such serine proteasesinclude trypsin, chymotrypsin, pancreatic elastase II, cathepsin G,prostate-specific antigen, leukocyte elastase, chymase, tryptase,acrosin, human tissue kallikrein, and plasmin. Plasmin includesGlu-plasmin, Lys-plasmin, derivatives and modified or truncated variantsthereof, including, but not limited to, midi-plasmin, mini-plasmin, ormicro-plasmin.

The methods of the present invention comprise acidic conditions andstabilizers that render the plasmin stable for storage. The plasmin usedin the present invention is obtained by activating plasminogen, and isisolated and stored in an acidified solution having a pH of less thanabout 4 to provide a stable formulation of plasmin. The fibrinolyticcomposition of the present invention may be lyophilised and issubstantially free of plasminogen activator. The low buffering capacityof the plasmin composition allows rapid titration to a physiological pHwhen administered to a thrombus or serum. Acidified plasmin is rapidlyneutralized and activated when administered in a low buffering capacitysolution directly to the clot site for thrombolytic therapy.

Additional objects, features, and advantages of the invention willbecome more apparent upon review of the detailed description set forthbelow when taken in conjunction with the accompanying drawing figures,which are briefly described as follows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the pH dependence of plasmin activity as measuredwith the chromogenic substrate S2251.

FIG. 2 illustrates plasmin stability in acidified water (pH 3.7) asmeasured by a caseinolytic assay.

FIG. 3 illustrates a plot of pH versus percent heavy chain relative tototal protein in each lane of SDS gels.

FIG. 4 illustrates the effectiveness of plasmin or tPA plus plasminogenin thrombolysis.

FIG. 5 illustrates thrombolytic potency of active plasmin.

FIG. 6 illustrates the effect of plasma inhibitors on plasmin-inducedthrombolysis.

FIG. 7 compares local administration of tPA or Lys-plasmin on blood flowrestoration, and FVIII and fibrinogen consumption, at 60 minutes.

FIG. 8 compares local administration of tPA or Lys-plasmin on blood flowrestoration at 90 minutes, and FVIII and fibrinogen consumption at 60minutes.

FIG. 9 illustrates the effect of local administration of tPA versusplasmin on cuticle bleed times.

FIG. 10 illustrates lysis of retracted blood clots with equimolar amountof reversibly inactivated acidified plasmin or mini-plasmin.

FIG. 11 compares local administration of tPA and plasmin on thrombuslysis, and Factor VIII and fibrinogen consumption, at 60 minutes.

FIG. 12 compares local administration of tPA and plasmin on thrombuslysis at 90 minutes, and Factor VIII and fibrinogen consumption at 60minutes.

FIG. 13 illustrates the extent of clot lysis by tPA and reversiblyinactivated acidified plasmin as a PAO model.

FIG. 14 illustrates the progressive degradation of a plasmin compositionat a pH of 2.2, 3.5, or 3.7.

FIG. 15 illustrates the cleavage sites generated in plasmin at pH 2.2and 3.8.

FIG. 16 illustrates the stability at 37° C. of a reversibly inactivatedacidified plasmin at pH of 3.7, with carbohydrate stabilizers.

FIG. 17 illustrates the stability at 37° C. of a reversibly inactivatedacidified plasmin at a pH of 3.7 with glucosamine, niacinamide, thiamineor citrulline as a stabilizing agent.

FIG. 18 illustrates the titration, with human serum, of plasminsolutions having various low buffering capacity buffers.

DETAILED DESCRIPTION OF THE INVENTION

A full and enabling disclosure of the present invention, including thebest mode known to the inventors of carrying out the invention, is setforth more particularly in the remainder of the specification, includingreference to the Examples. This description is made for the purpose ofillustrating the general principles of the invention and should not betaken in the limiting sense.

The present invention addresses the need for a method of thrombolysisthat allows the use of a fibrinolytic composition comprising reversiblyinactivated acidified plasmin and the localized delivery of the plasminto a thrombotic occlusion. The invention provides a method foradministering a therapeutic dose of a fibrinolytic composition to ahuman or animal having a thrombotic occlusion, comprising administeringparenterally to the human or animal a therapeutic dose of apharmaceutically acceptable reversibly inactivated acidifiedfibrinolytic composition substantially free of plasminogen activator,allowing the administered fibrinolytic composition to interact with thethrombotic occlusion, monitoring the level of vascular flow of the humanor animal, and repeating these steps until a pre-selected level ofvascular flow is attained. The method of the invention further providesfor the use of a fibrinolytic composition comprising an reversiblyinactivated acidified plasmin substantially free of plasminogenactivator in a low buffering capacity buffer. The method also providesfor the intravascular catheter direct delivery of the fibrinolyticcomposition into or in the immediate vicinity of the thrombus, therebyminimizing the systemic degradation of fibrin while retaining themaximum plasmin activity against the thrombus.

With the escalating use of arterial and venous catheters in the clinics,local delivery of reversibly inactivated acidified plasmin in closeproximity to, or actually into, a thrombus offers an attractivetherapeutic opportunity for thrombolysis. Being an active serineprotease, plasmin is a direct thrombus dissolving agent, in contrast toplasminogen activators that require the presence of the zymogenplasminogen in the vicinity of the thrombus. Local catheter-directedthrombolytic therapy with active plasmin can be regulated to achievelocalized total thrombolysis, and plasmin has the potential to be asafer thrombolytic agent because the lower dosage required for localdelivery may significantly reduce bleeding complications frequentlyassociated with high dose thrombolytic therapy induced by plasminogenactivators. Any spillage of plasmin from the immediate vicinity of thethrombus site will be rapidly neutralized by circulating α₂-antiplasmin.

There are several technical challenges associated with plasminpurification, and storage, as well as with its therapeutic use anddelivery. Plasmin is an active serine protease and is subject toautodigestion and inactivation at physiological pH. Plasmin degradation,unfortunately, is also most evident in the pH range required for in vivothrombolysis.

The fibrinolytic composition, as incorporated into the presentinvention, includes the maintenance of the plasmin in an acidic bufferduring purification, as well as its formulation in an acidified carrierhaving a pharmaceutically acceptable low buffering capacity buffer,thereby providing a reversibly inactivated acidified plasmin-containingfibrinolytic composition substantially free of plasminogen activator. Itis contemplated to be within the scope of the present invention for thefibrinolytic composition to be a lyophilized composition that may bereconstituted by the addition of a pharmaceutically acceptable carriersuch as, but not limited to, water, physiological saline or any othersolvent that will allow administration of the composition to a human oranimal. Its efficacy in restoring vascular patency was demonstrated inin vitro assays and in an in vivo rabbit jugular vein thrombolysismodel.

The term “reversibly inactivated” as used herein refers to an enzymicactivity that is substantially free of activity under a specific set ofconditions but will revert to an active form when transferred to anotherset of conditions.

The term “pharmaceutically acceptable carrier” as used herein refers toany carrier that is physiologically tolerated by a recipient human oranimal, including, but not limited to, water, salt solutions,physiological saline, or any other liquid or gel in which a fibrinolyticagent such as plasmin may be dissolved or suspended. The“pharmaceutically acceptable carrier” may include any pharmaceuticallyacceptable compound that will give a plasmin solution having a pH belowabout 4.0 and which has low or zero buffering capacity.

The term “low buffering capacity buffer” as used herein refers to theamount of acid or base that a buffer can neutralize before the pH beginsto change to an appreciable degree. As used herein a low bufferingcapacity buffer will be significantly pH adjusted by the addition of asmall volume of an acid or base relative to the volume of the lowbuffering capacity buffer solution. This term is meant to includesolutions acidified by strong acids including, but not limited to,hydrochloric acid, nitric acid or sulfuric acid, and which have nobuffering capacity.

The term “physiological pH” as used herein refers to a pH between aboutpH 6.5 and 7.5, more typically between pH 7.1 to 7.5.

The term “thrombus” as used herein refers to a thrombus in a bloodvessel or a device contacting blood (e.g. catheter devices or shunts). Athrombus may comprise fibrin and may further comprise, but is notlimited to, platelets, erythrocytes, lymphocytes, lipid, serumconstituents or any combination thereof. A “thrombus” may be, but is notlimited to, an annular thrombus, ball thrombus, hyaline thrombus, muralthrombus, stratified thrombus or white thrombus.

The term “thrombotic occlusion” as used herein refers to a partial ortotal blockage of a vessel due to the formation of a thrombotic clot,wherein the thrombus comprises at least fibrin. The vascular vesseloccluded may be, but is not limited to, a vein, artery, venule,arteriole, capillary, vascular bed or the heart and may be within anyvascularized organ or tissue of the human or animal body. The thromboticocclusion may also be of a catheter or other implant, including, but notlimited to, prosthetic vessels and grafts of synthetic, human or animalorigin, effectively blocked by an occlusion comprising fibrin.

The term “catheter device” as used herein refers to any catheter ortube-like device that may enter the body, and includes but is notlimited to, an arterial catheter, cardiac catheter, central catheter,central venous catheter, intravenous catheter, balloon catheter devicesperipherally inserted central catheter, pulmonary artery catheter ortunneled central venous catheter and arterio-venal shunts.

The term “pharmaceutically acceptable acidified carrier” as used hereinrefers to any pharmaceutically acceptable carrier that has beenacidified to a pH below about 4.0. The “pharmaceutically acceptableacidified carrier” may comprise a low or zero buffering capacity buffersuch as a carboxylic acid such as, but not limited to, formic, acetic,proprionic, butyric, citric, succinic, lactic or malic acids acidifiedto a pH below about 4.0 by the addition of an inorganic acid; or atleast one amino acid such as, but not limited to, glycine, alanine,valine, isoleucine, threonine or glutamine, or at least one inorganicacid such as, but not limited to, sulfuric acid, hydrochloric acid,nitric acid or phosphoric acid or any combination thereof. It iscontemplated to be within the scope of the present invention for theacid moiety of the pharmaceutical carrier to be at least onephysiologically tolerated buffer, oligopeptide, inorganic or organic ionor any combination thereof that will maintain a pH in thepharmaceutically acceptable carrier below a value of about 4.0.

The term “carbohydrate” as used herein refers to any pharmaceuticallyacceptable saccharide or disaccharide such as, but not limited to,glucose, fructose, maltose or mannose, sugar alcohols including, but notlimited to, sorbitol and mannitol, and polysaccharides such as, but notlimited to, dextrins, dextrans, glycogen, starches and celluloses, orany combination or derivative thereof that are pharmaceuticallyacceptable to a human or animal.

The term “stabilizing agent” as used herein refers to at least onecompound such as, but not limited to, glycerol, ascorbate, niacinamide,glucosamine, thiamine or inorganic salt such as, but not limited to,sodium chloride, potassium chloride, magnesium chloride or manganesechloride or any combination thereof that will increase the stability ofa preparation of plasmin.

The term “reversibly inactivated acidified plasmin” as used hereinrefers to any catalytically active form of plasmin capable ofproteolytically cleaving fibrin when under physiological conditions, butreversibly inactivated when placed at a pH between about pH 2.5 to about4.0. The term “inactivated” as used herein refers to a total orsubstantial reduction in enzymatic activity compared to the activity atphysiological pH. The term “active plasmin” as used herein refers to aplasmin under conditions where the plasmin is capable of proteolyticallycleaving fibrin. The term “plasmin” includes, but is not limited toGlu-plasmin, Lys-plasmin, derivatives, modified or truncated variantsthereof. The term “truncated variants” includes, but is not limited to,the midi-plasmin, mini-plasmin or micro-plasmin as disclosed in U.S.Pat. No. 4,774,087 incorporated herein by reference in its entirety.

The term “anti-coagulant” as used herein refers to any compound capableof inhibiting the formation of a thrombus including, but not limited to,hiruidin, heparin, thrombin inhibitors, platelet inhibitors, plateletaggregation inhibitors and any derivatives or combinations thereof.

The term “serine protease” as used herein refers to any serine proteasecapable of proteoytically cleaving fibrin including, but not limited to,plasmin, trypsin, chymotrypsin, pancreatic elastase II, cathepsin G,prostate-specific antigen, leukocyte elastase, chymase, tryptase,acrosin and human tissue kallikrein.

One limitation of current thrombolytic therapy with plasminogenactivators is plasminogen availability surrounding or within a thrombus.The local delivery of a fibrinolytic agent to a thrombus now allowsplasmin itself to be a potent therapeutic agent directly administered toa thrombus. In contrast to various plasminogen activators that arecurrently used as thrombolytics, direct localized thrombolytic therapywith plasmin can be intensified to whatever level is required to achieveclot lysis. This is because plasmin acts directly upon the fibrinpolymer. Also, plasmin, when delivered directly into or adjacent to athrombus, allows a lower effective dose to be administered with aconcomittant reduction in the systemic hemorrhage typically associatedwith conventional thrombolytic therapy. Excess plasmin can also berapidly inactivated by circulating α₂-antiplasmin.

Highly purified active plasmin of the present invention was thereforeprepared from plasminogen that had been purified from Cohn FractionII+III. The purity of plasmin was greater than 95% and specific activitywas in the range of 18-23 CU/mg. The plasmin preparations weresubstantially free of urokinase, or any other plasminogen activator,that had been used for the conversion of plasminogen into plasmin. Thepresent invention also contemplates that plasmin substantially free ofplasminogen activator may be prepared from any source including, but notlimited to, mammalian serum, a recombinant plasminogen or a truncatedplasminogen such as, but not limited to, the mini-plasminogen ormicro-plasminogen as disclosed by Wu et al. in U.S. Pat. No. 4,774,087,incorporated herein by reference in its entirety.

The plasmin of the present invention was purified to substantiallyremove plasminogen activators by binding to a benzmidine-Sepharosechromatography column and eluted plasmin was collected and stored in anacidified pharmaceutically acceptable carrier having a low bufferingcapacity buffer. A low pH in the range of about 2.5 to about 4.0significantly stabilized the plasmin, even when held at room temperatureor greater. While not bound by any one theory, it is believed that atthis low pH value the plasmin no longer has serine protease activitythat would otherwise lead to autodegradation of the plasmin, as would beseen when plasmin is to be stored at the physiological pH of about7.0-7.5.

When the reversibly inactivated acidified plasmin is administered,according to the methods of the present invention, directly into athrombus or proximal thereto, the reversibly inactivated acidifiedplasmin in the low or zero buffering capacity buffer encounters theserum buffering capacity at the physiological pH of about 7.4. The lowbuffering capacity of the pharmaceutically acceptable carrier isneutralized, whereupon the plasmin reverts to its active state andproteolytically digests the fibrin of the thrombus.

By initially employing a method of plasmin preparation that rendersplasmin proteolytically inactive until administered into, or immediatelyadjacent to, a thrombus, and which is also substantially free of anyplasminogen activator, the likelihood of inducing undesirable systemichemorrhage is diminished. Excess administered plasmin is rapidlyinactivated by circulating serum inhibitors such as α₂-antiplasmin, andthe relatively stable plasminogen activators that would otherwisecirculate to induce distal fibrinolysis are substantially absent.

Acidified plasmin may be readily stored in pharmaceutically acceptablecarriers having a low or zero buffering capacity such as, but notlimited to, aqueous 5 mM glycine. Any pharmaceutically acceptable ionmay be used, singularly or in combination, if a pH is in the range ofabout 2.5-4.0. Typically, reversibly inactivated acidified plasmin hasbeen maintained at a pH of 3.1-3.5. The pharmaceutically acceptableacidic ions incorporated in the carrier to maintain a low pH may beselected from oligopeptides, at least one amino acid, or organic orinorganic acids or a combination thereof. Stabilization may be furtherenhanced by the inclusion of at least one pharmaceutically acceptablecompound that may be, but is not limited to a carbohydrate.

A description of the method of treating a thrombotic occlusion in apatient using a therapeutically effective dose of the low-pH plasmincompositions of the present invention is disclosed in U.S. patentapplication Ser. No. 10/143,112, entitled “Reversibly InactivatedAcidified Plasmin”, commonly assigned and filed contemporaneously withthe instant application, and is incorporated herein by reference in itsentirety.

Additionally, a process for producing the reversibly inactivatedacidified plasmin composition of the instant invention is disclosed inU.S. patent applicatio Ser. No. 10/143,156, entitled “Process for theProduction of a Reversibly Inactivated Plasmin Composition”, commonlyassigned and filed contemporaneously with the instant application, andis incorporated herein by reference in its entirety.

The administration of the plasmin composition can be by any method thatwill deliver the plasmin as a bolus or as a prolonged infusion directlyinto a thrombus, or to a site only a short distance proximal to thethrombus, whereupon it can rapidly encounter the thrombus. By minimizingthis distance to the thrombus or by directly administering into theclots, plasmin exposure to plasmin inhibitors in the serum lying betweenthe delivery needle or catheter and the thrombus is reduced. The presentinvention contemplates that the reversibly inactivated acidified plasminmay be delivered to the thrombus by a catheter. The present inventionfurther contemplates that a cannulated needle, such as, but not limitedto a syringe needle, may be used to inject the reversibly inactivatedacidified plasmin into a thrombus. It is, however, within the scope ofthe present invention for any means known to one of skill in the art, tolocally administer a fluid to a specific location in a human or animal.Catheter delivery to a vascular or coronary thrombus further allowsprecision in placing the plasmin, especially within the thrombus. Whilethe present invention provides methods of localized delivery of anreversibly inactivated acidified plasmin in a low buffering capacitybuffer to a thrombus, it is within the scope of the present inventionfor delivery of the reversibly inactivated acidified plasmin compositionthereof to a fibrin occlusion of a catheter implanted in a human oranimal.

Using an in vitro ¹²⁵I-fibrin-labeled thrombolysis assay, it was shownthat plasmin was capable of dissolving thrombi in a dose-dependentmanner. Fibrinolysis was enhanced by deletion of either α₂-antiplasminor α₂-microglobulin in plasma surrounding the thrombus, as well as bythe deletion of all inhibitors (i.e., in PBS). These results show thatthrombolysis by plasmin is under very strict regulation by theendogenous plasmin inhibitors, and provides the basis for saferthrombolytic therapy.

The in vivo efficacy of reversibly inactivated acidified plasmin (1-3mg/kg) in a low buffering capacity buffer directly administered locallyto a thrombus via a catheter was compared with that of tPA (0.5 and 1.0mg/kg) in the rabbit jugular vein thrombosis model. The rate ofthrombolysis was monitored. In addition, the consumption of Factor VIIIand fibrinogen, as well as cuticle bleeding time (CBT), were measured asindicators of the systemic lytic state. In comparison with 0.5 mg/kg oftPA, plasmin provided comparable or significantly better thrombolysis,with comparable consumption of Factor VIII and fibrinogen and similarCBT at 1 mg/kg dose. There was a significantly increased consumption andCBT with plasmin at 3 mg/kg. When compared with 1 mg/kg of tPA, bothplasmin and tPA provided very similar thrombolysis rates withsignificantly less consumption of fibrinogen at 3 mg/kg plasmin.

Thus, reversibly inactivated acidified plasmin can be effectively andsafely stored and used as a thrombolytic agent during catheter-assistedlocal thrombolysis without neutralization before administration to ahuman or animal. Plasmin has comparable, if not superior, fibrinolyticactivity compared to tPA, and the safety profile appears at leastsimilar in this animal model of local thrombolytic delivery.

It is contemplated to be within the scope of the present invention thatthe reversibly inactivated acidified fibrinolytic enzyme may be, but isnot limited to, plasmin, derivatives of plasmin such as truncated formsthereof including, but not limited to, mini-plasmin and micro-plasmin.

The present invention provides a method of administering a therapeuticdose of a fluid fibrinolytic composition preparation substantially freeof plasminogen activators to the immediate vicinity of, or directlyinto, a thrombotic occlusion of a vascular vessel or an occludedcatheter device. The invention further provides a method for deliveringa therapeutic dose of a fluid reversibly inactivated acidified plasmincomposition to a vascular thrombotic occlusion wherein the plasmin maybe stabilized prior to administration at a pH of about 4.0. The presentinvention further provides a method for delivering a therapeutic dose ofa fluid reversibly inactivated acidified plasmin in a low bufferingcapacity buffer directly into a vascular thrombotic occlusion, orthrombus, whereupon the plasmin composition reverts to a physiologicalpH that allows the plasmin to proteolytically cleave fibrin.

In one embodiment of the present invention, the method of the inventioncomprises the steps of identifying a human or animal having a thromboticocclusion, administering to the human or animal a therapeutic dose of apharmaceutically acceptable reversibly inactivated acidifiedfibrinolytic composition in a low buffering capacity buffer, allowingthe administered fibrinolytic composition to interact with the vascularocclusion, monitoring the level of vascular flow of the human or animal;and repeating steps of administering the fibrinolytic composition untila pre-selected level of vascular flow is attained.

In another embodiment of the present invention, the reversiblyinactivated acidified fibrinolytic composition comprises plasmin and apharmaceutically acceptable acidified carrier having a low bufferingcapacity buffer.

In still another embodiment of the present invention, thepharmaceutically acceptable acidified carrier comprises water and atleast one pharmaceutically acceptable acid, wherein the acid is anorganic acid, which may be, but is not limited to, a carboxylic acid, anoligopeptide, an amino acid, or an inorganic acid and having a low orzero buffering capacity, and the acidified carrier has a pH less thanabout 4.0.

In yet another embodiment, the pharmaceutically acceptable acidifiedcarrier comprises water and a pharmaceutically acceptable acid selectedfrom the acid forms of formate, acetate, citrate, glycine, isoleucine,serine, threonine, glutamine, and alanine, wherein the acidified carrierhas a pH less than about 4.0.

In a further embodiment, the pharmaceutically acceptable acid is theacid form of acetate or citrate.

In yet another embodiment, the pharmaceutically acceptable acid is theacid form of acetate.

In an embodiment of the method of the present invention, the reversiblyinactivated acidified fibrinolytic composition has a pH of between about2.5 and about 4.0. In another embodiment of the method of the presentinvention, the fibrinolytic composition has a pH of about 3.7.

In other embodiments of the method of the present invention, the plasminis in the concentration range of between about 0.01 mg/ml to about 50mg/ml. In one embodiment of the method of the present invention theplasmin is in the concentration range of between about 0.1 mg/ml toabout 10 mg/ml.

In further embodiments of the method of the present invention, theacidic ion is in the concentration range of between about 1 mM to about500 mM. In one embodiment of the method of the present invention, theacidic ion is in the concentration range of between about 1 mM and 50mM.

Embodiments for practicing the method of the present invention furthercomprise a physiologically acceptable plasmin stabilizer, wherein thecarbohydrate is selected from glucose, maltose, mannitol, sorbitol,sucrose, lactose or trehalose, wherein the sugar has a concentration inthe range of about 0.2% w/v to about 20% w/v. In one embodiment, thesugar is glucose and the concentration thereof is about 20%.

Embodiments of the method of the present invention further comprise thetherapeutic dose of the reversibly inactivated acidified plasmin in therange of between about 0.01 mg plasmin/kg body weight and 10 mgplasmin/kg body weight.

In embodiments of the method of the present invention, the reversiblyinactivated acidified fibrinolytic composition is administeredintravascularly or intrathrombus. In one embodiment of the method of thepresent invention, the reversibly inactivated acidified plasmin in a lowpH-buffered capacity buffer is administered directly into, or proximallyto a thrombus by an intravascular catheter device. In still anotherembodiment of the method of the present invention, the intravascularcatheter is directed to a vascular occlusion.

Even though the invention has been described with a certain degree ofparticularity, it is evident that many alternatives, modifications, andvariations will be apparent to those skilled in the art in light of thepresent disclosure. Accordingly, it is intended that all suchalternatives, modifications, and variations that fall within the spiritand the scope of the invention be embraced by the defined claims.

The following examples are presented to describe preferred embodimentsand utilities of the present invention, but should not be construed aslimiting thereof.

It should be appreciated by those of skill in the art that thetechniques disclosed in the examples that follow represent techniquesdiscovered by the inventors to function well in the practice of theinvention, and thus can be considered to constitute preferred modes forits practice. Those of skill in the art, in light of the presentdisclosure, will appreciate that many changes can be made in thespecific embodiments disclosed and still obtain like or similar resultswithout departing, again, from the spirit and scope of the presentinvention.

EXAMPLE 1 Sources of Proteins Investigated

Plasminogen was purified from Cohn Fraction II+III paste by affinitychromatography on Lys-Sepharose as described by Deutsch & Mertz (1970).Thus, 200 g of the paste was resuspended in 2 liter of 0.15M sodiumcitrate buffer, pH 7.8. The suspension was incubated overnight at 37°C., centrifuged at 14,000 rpm, filtered through fiberglass and mixedwith 500 ml of Lys-Sepharose 4B (Pharmacia). Binding of plasminogen wasat room temperature for 2 hours. The Lys-Sepharose was then transferredonto a 2-liter glass filter, and washed several times with 0.15M sodiumcitrate containing 0.3M NaCl until the absorbance at 280 nm droppedbelow 0.05. Bound plasminogen was eluted with three 200-ml portions of0.2M ε-aminocaproic acid. Eluted plasminogen was precipitated with 0.4 gsolid ammonium sulfate/ml of plasminogen solution. The precipitate ofcrude (80-85% pure) plasminogen was stored at 4° C.

Low-molecular weight urokinase (LMW-urokinase) (Abbokinase-AbbottLaboratories, Chicago Ill.) was further purified by affinitychromatography on benzamidine-Sepharose. The urokinase was then coupledto CNBr-activated Sepharose 4B by mixing 1.3 mg of LMW-urokinase in 50mM acetate buffer, pH 4.5, and diluting with 5 ml of the couplingbuffer, 0.1M sodium bicarbonate, pH 8.0.

This solution was immediately combined with 5 ml of CNBr-activatedSepharose previously swollen and washed in 0.1M HCl. The couplingoccurred for 4 hours on ice with shaking. The excess of the CNBr activegroup was blocked with 0.1M Tris, pH 8.0. Each batch ofurokinase-Sepharose was used 5 times and stored in 50% glycerol in waterat 4° C. between the cycles. Tissue plasminogen activator (ACTIVASE™)was from Genentech. Plasminogen-free fibrinogen and α-thrombin (3793U/ml) were from Enzyme Research, Inc. α₂-Antiplasmin was obtained fromAthens Research Technologies. Commercially available plasmin was fromHaemotologic Technologies, Inc. Chromogenic plasmin substrate S2251 wasfrom Chromogenix. ¹²⁵I-Labeled human fibrinogen (150-250□Ci/mg) was fromAmersham Pharmacia Biotech. SDS-polyacrylamide gel electrophoresis wasperformed in the Pharmacia Phast System apparatus using pre-made 8-25%gradient gels and SDS-buffer strips.

EXAMPLE 2 Purification of Active Plasmin

(i) Activation of Plasminogen to Plasmin Using Urokinase-Sepharose

Plasminogen was cleaved to plasmin yielding plasmin withoutcontamination of the final preparation by using an immobilizedplasminogen activator. Urokinase cleaves plasminogen directly.Plasminogen activation by urokinase does not depend on the presence offibrin as in the case of tPA, and urokinase is a human protein. Thesefactors, and its relative low cost, make urokinase the preferredactivator, although this does not preclude the use of tPA, streptokinaseor any other cleavage means yielding an active plasmin capable of fibrindegradation. The ammonium sulfate precipitate of crude plasminogen wascentrifuged at 14,000 rpm and resuspended in a minimal volume using 40mM Tris, containing 10 mM lysine, 80 mM NaCl at pH 9.0 to achieve thefinal protein concentration of 10-15 mg/ml. The plasminogen solution wasdialyzed overnight against the same buffer to remove ammonium sulfate.The dialyzed plasminogen solution (10-20 ml) was diluted with an equalvolume of 100% glycerol and combined with 5 ml of urokinase-Sepharose.The use of 50% glycerol reduces autodegradation of plasmin formed duringactivation. Plasmin is stable in 50% glycerol and can be stored in thissolution at −20° C. for an extended period.

The plasminogen activation occurred at room temperature for between 2hours and 24 hours depending on the freshness of theurokinase-Sepharose. With a fresh batch of urokinase-Sepharose,activation could be completed in 2 hours. It deteriorates, however, andbecomes less efficient after several cycles, necessitating the use ofSDS-PAGE under reducing conditions to monitor the progress ofplasminogen activation. Upon completion of the activation, the plasminsolution was filtered from the urokinase-Sepharose with a glass filter,and immediately applied to benzamidine-Sepharose.

(ii) Capturing of Plasmin on Benzamidine-Sepharose

Since the plasmin is a serine protease with trypsin-like specificity,benzamidine-Sepharose is an affinity absorbent that allowed capture ofthe active plasmin. A plasminogen solution in 50% glycerol was appliedto the 50 ml benzamidine-Sepharose column equilibrated with 0.05M Tris,pH 8.0, containing 0.5M NaCl with a flow rate of 3 ml/min. The columnwas run at 3 ml/min at 3-7° C. The front portion of the non-bound peakcontained high-molecular weight impurities. The rest of the non-boundpeak is represented by residual non-activated plasminogen and byinactive autodegradation products of plasmin.

(iii) Elution of the Bound Plasmin with Low-pH Buffer

To protect plasmin from inactivation at neutral pH conditions, acidicelution conditions were selected. The plasmin bound tobenzamidine-Sepharose was eluted with 0.2M glycine buffer, pH 3.0containing 0.5M NaCl. The bound peak was typically divided into threepools, two front peaks, B1 and B2, and the bulk of the eluted materialas B3.

Non-reducing gel analysis showed that all three pools contained highlypure (>95%) plasmin. The gel analysis, however, in addition to the heavyand light chains of plasmin, revealed some low molecular weight bands ina range of 10-15 kDa as a result of partial internal cleavagedegradation of the plasmin.

The front portion of peak B1 typically contained most of the lowmolecular weight impurities. The B2 and B3 pools were less degraded. Thefront portion of the bound peak had very little of the plasmin activityand was usually discarded. The loss of activity in this material may bedue to autodegradation during chromatography, because there is noglycerol present in the eluted material, and the pH of the front portionis intermediate between the pH of the equilibrating and eluting buffers,typically in a range of pH 6-6.5. The eluted plasmin, substantially freeof plasminogen activators, was collected in tubes containing 2M glycinebuffer, pH 3.0 (10% of the collected volume).

(iv) Formulation of Eluted Material in Acidified Water (pH 3.7)

Eluted plasmin was dialyzed against water that had been acidified toabout pH 3.7 with glacial acetic acid, or against water containing 0.15MNaCl also acidified to about pH 3.7 with glacial acetic acid.

Any acid providing a pharmaceutically acceptable acidified carrierhaving a low buffering capacity buffer and having a pH between about 2.5to about 4.0 can be used. For example, also contemplated within thescope of this invention is the use of other acids and amino acids suchas, but not limited to, inorganic acids, carboxylic acids, aliphaticacids and amino acids including, but not limited to, formic acid, aceticacid, citric acid, lactic acid, malic acid, tartaric acid, benzoic acid,serine, threonine, valine, glycine, glutamine, isoleucine, β-alanine andderivatives thereof, either singly or any combination thereof, that willmaintain the pH in the pharmaceutically acceptable carrier of about 2.5to about 4.0.

Plasmin is extremely stable in acidified water and can be effectivelyused in this form for in vitro and in vivo studies. Plasmin specificactivity was measured using an adapted caseinolytic assay as describedby Robbins & Summaria (1970). One ml of 4% casein solution in acidifiedwater and an appropriate volume of 67 mM sodium phosphate buffer, pH 7.4was added to a test polycarbonate tube. The solutions were vortexed andincubated at 37° C. for 10 minutes. Plasmin samples or buffer (blank)were added to each tube at 15-second intervals, mixed thoroughly andincubated at 37° C. for 30 minutes. The reaction was stopped with theaddition of 3 ml of 15% trichloroacetic acid and the precipitate wasallowed to form for 15 minutes. The tubes were centrifuged at 3200 rpmfor 20 minutes. The supernatants were transferred to cuvettes and theA₂₈₀ of each sample was determined. The specific caseinolytic activityof each sample was determined by the following formula:$\frac{3.27 \times \left\lbrack {{A_{280}\left( {{Plasmin}\quad{Sample}} \right)} - {A_{280}({Blank})}} \right\rbrack}{{µg}\quad{Plasmin}\quad{in}\quad{Assay}} = \begin{matrix}{{caseinolytic}\quad{units}} \\{({CU})\text{/}{mg}\quad{protein}}\end{matrix}$The plasmin concentration was determined spectrophotometrically usingthe extinction coefficient of 1.7 for 0.1% solution.

EXAMPLE 3 pH-Dependent Stability of Plasmin

Plasmin exhibits a bell-shaped pH dependence of its catalytic activity.As shown in FIG. 1, plasmin has maximum enzyme activity at pH 7.5-8.0,and its activity rapidly decreases at either more alkaline or moreacidic pH values. Plasmin is most inactive, and reversibly so, below pH4.0, due to the protonation of histidine in the catalytic center, asshown by Robbins & Summaria, (1976) and Castellino & Powell (1981).

Plasmin is very unstable at a physiological pH. Both the heavy chain andlight chains of plasmin degraded dramatically within hours at roomtemperature and 4° C. Plasmin was formulated at 1 mg/ml in 0.04M sodiumphosphate, pH 7.4, and incubated at 22° C. or 4° C. for 6 hours. Duringthe incubation, the plasmin integrity was analyzed every two hours byreducing SDS-PAGE analysis. Both the heavy chain and light chaindegraded rapidly within hours at 22° C. and 4° C. as shown in Table 1.

TABLE 1 The rapid degradation of plasmin in neutral pH solution at 22°C. and 4° C. % of intact heavy chain % of intact light chain PlasminBuffer pH Temp Initial 2 hr 4 hr 6 hr Initial 2 hr 4 hr 6 hr 1 mg/ml0.04 M 7.4 22° C. 100% 27% 27% 29% 100% 29% 26% 28% PO₄ 1 mg/ml 0.04 M7.4  4° C. 100% 32% 27% 25% 100% 33% 25% 22% PO₄ (The intact heavy chainand light chain of plasmin at initial time point were normalized as100%.)

Plasmin at 1 mg/ml was incubated at 37° C. for 14 days under differentacidic conditions. The changes in plasmin heavy chain and light chainwere analyzed by reducing SDS-PAGE. Plasmin was formulated at 1 mg/ml in0.04M sodium phosphate, pH 7.4 and was also incubated at 4° C. for sixhours. During the incubation, the activity of the plasmin sample wasmeasured every two hours by chromogenic potency assay. Plasmin potencywas quantitatively measured using the MLA 1600C analyzer (Pleasantville,N.Y.). Plasmin hydrolyzed the chromogenic substrate S-2403(D-pyroglutamyl-L-Phenylalanyl-L-Lysine-p-Nitroaniline hydrochloride orabbreviated as pyro-Glu-Phe-Lys-pNA) to form peptide and thechromophoric group p-nitroaniline (pNA). The rate of color formation wasmeasured kinetically at 405 nm. The amount of substrate hydrolyzed wasproportional to the plasmin activity in the sample. A standard curve wasgenerated from the linear regression of the rate of color formation(OD/min) versus the potency of a plasmin standard. The linear equationtogether with the observed rate for an unknown sample was used tocalculate the potency of unknowns. The potency of plasmin was reportedin units of mg/ml.

Plasmin integrity was significantly decreased by incubation at aphysiological pH, as shown in Table 2.

TABLE 2 The rapid decrease of plasmin activity in neutral pH solution at4° C. Chromogenic Potency Plasmin Buffer pH Initial 2 hr 4 hr 6 hr 1mg/ml PO₄, 0.04 M 7.4 100% 43.3% 32.6% 26.4% (*The activity of plasminsolution at initial time point was normalized as 100%.)

In this neutral pH solution, plasmin activity decreased more than 70%after 6 hours at

Plasmin formulated in acidified water at pH 3.7 is stable. It can bekept in this form for months at reduced temperatures without any loss ofactivity or the appearance of degradation products of a proteolytic oracidic nature. FIG. 2 and the data of Table 3 show the stability ofplasmin at 4° C. and at room temperature.

TABLE 3 Stability of 1 mg/ml plasmin in the following acidic conditionsat 37° C. % intact heavy chain % intact light chain Plasmin after 14days after 14 days Formulation (mg/ml) Acidic Condition pH at 37° C. at37° C.  1 1 5 mM HAC/NaAc 2.5  19%  62%  2 1 5 mM HAC/NaAc 3.0  41%  92% 3 1 5 mM HAC/NaAc 3.4  48%  92%  4 1 5 mM HAC/NaAc 3.4  49%  96%  5 1 5mM HAC/NaAc 3.4  50%  96%  6 1 5 mM HAC/NaAc 3.7  13% 123%  7 1 5 mMHAC/NaAc 4.0 9.3% 107%  8 1 5 mM citric 2.27 9.3%  64% acid/Na citrate 9 1 5 mM citric 3.1  33%  68% acid/Na citrate 10 1 5 mM citric 3.56 46%  88% acid/Na citrate 11 1 5 mM citric 4.0 7.4% 104% acid/Na citrate12 1 5 mM glycine 2.2 7.3% 104% 13 1 5 mM glycine 3.1  36%  70% 14 1 5mM glycine 3.5  49%  85% 15 1 5 mM glycine 3.8  12%  85% 16 1 5 mMglycine 4.1   6%  81% 17 1 5 mM serine 3.4  56% 100% 18 1 5 mM threonine3.4  54% 100% 19 1 5 mM valine 3.4  52%  96% 20 1 5 mM isoleucine 3.4 51% 100% 21 1 5 mM β-alanine 3.7  33%  90% 22 1 2 mM benzoic 3.5  42% 93% acid 23 1 2 mM lactic acid 3.5  45%  91% 24 1 2 mM malic acid 3.5 50%  90% 25 1 2 mM tartaric acid 3.5  28%  87% (The intact heavy chainand light chain in each formulation before incubation were normalized as100%; HAc/NaAc = acetic acid/sodium acetate)

At 4° C., plasmin is stable for at least nine months. Even at roomtemperature, reversibly inactivated acidified plasmin is stable for atleast two months. Long-term stability at room temperature is importantbecause it would make this formulation compatible with long regimens ofthrombolytic administration. For example, 36 hour administration ofthrombolytics such as tissue plasminogen activator or urokinase iscommon in treatment of peripheral arterial occlusions. Shown in FIG. 3is a plot of pH versus percent heavy chain relative to total protein ineach lane of the SDS gels. The results demonstrate a pH stabilityoptimum of about 3.1-3.5, irrespective of the type of buffer, or bufferconcentration.

The ability of reversibly inactivated acidified plasmin to become fullyactive upon transfer to physiological pH is evidenced by its activity inthe caseinolytic assay and also in the ¹²⁵I-fibrin-labeled thrombolysisassays. Both of these assays are performed at pH 7.4, and there wascomplete recovery of plasmin activity during the change of pH andpassing through the isoelectric point (pH 5-5.5). The plasmin isformulated in a low-buffering capacity solvent and, when added to abuffered solution such as plasma, it rapidly adopts the neutral orphysiological pH instantly and the precipitation that usuallyaccompanies the slow passage through the isoelectric point, does notoccur.

EXAMPLE 4 Plasmin has the Same Intrinsic Fibrinolytic Potency as aPlasminogen/Plasminogen Activator Mixture

Plasmin has the same intrinsic fibrinolytic potency as aplasminogen/plasminogen activator mixture. Fibrinolytic potency ofplasmin was compared with that of a Lys-plasminogen and tPA mixture.These experiments were performed in a defined system consisting of an¹²⁵I-radiolabeled fibrin thrombus submersed in PBS. FIG. 4 shows that,in a buffered environment, thrombolysis achieved with plasmin is almostidentical to the Lys-plasminogen plus tPA mixture (curves a and b,respectively). At the same time, no thrombolysis was observed with tPAalone (curve c) or in the absence of any proteins (curve d). The dataobtained with tPA alone shows that its activity is dependent on itssubstrate, plasminogen, to be an effective thrombolytic.

These data indicate that, in the absence of inhibitors and other proteinfactors present in plasma, there is no difference in the ability to lysefibrin thrombi between purified plasmin and the combination of tPA andLys-plasminogen activated with tPA. In order to assess the thrombolyticpotency of active plasmin, the ¹²⁵I-fibrin-labeled thrombolysis assaywas performed with plasma thrombi in a plasma environment.

EXAMPLE 5 Role of Plasmin Inhibitors in Regulating Plasmin Activity

¹²⁵I-fibrin labeled thrombolysis assay. The fibrinolytic properties ofplasmin were determined in an in vitro system consisting of aradio-labeled plasma thrombus immersed in human citrated plasma asdescribed by Lijnen et al. (1986). Plasma used in all experiments wassingle donor plasma thawed at 37° C., aliquoted, re-frozen and stored at−80° C. The stock solution of ¹²⁵I-labeled fibrinogen was prepared byrehydrating the contents of the vial (approximately 110 μCi/vial) with1.0 ml of 0.15M sodium citrate and was stored at 4° C.

Ten μl of ¹²⁵I-fibrinogen was added to a polycarbonate test tubecontaining 250 μl of plasma at 37° C. and mixed briefly. Twenty-five μlof a-thrombin, diluted with 0.1M CaCl₂ to a final concentration of 10-20μM, was added to the plasma. The radio-labeled thrombi were allowed toage for five minutes at 37° C. and then washed with PBS. The thrombiwere transferred into test tubes containing 2.25 ml plasma or buffer,one thrombus per tube.

A baseline radioactivity sample was measured for each thrombus. Plasminwas added to each tube after the addition of each thrombus. The thrombiwere returned to 37° C. for the duration of the experiment. Furthersamples were taken at indicated time points for measurement of releasedradioactivity. The extent of thrombolysis was calculated from the ratiobetween the amount of radioactivity released from the thrombus into theplasma and the total amount of soluble radioactivity in the reactiontube. The release of labeled fibrin degradation products, expressed inpercent, was plotted versus time.

In addition to the plasma milieu, some thrombolysis experiments wereconducted in a buffer environment or with plasma lacking α₂-antiplasminor α₂-macroglobulin activity. α₂-antiplasmin-depleted plasma wasobtained by passing normal plasma through a Kringles 1-3-Sepharosecolumn as described by Wiman (1980). α₂-Macroglobulin-inactivated plasmawas obtained by treatment of normal plasma with 0.1M methylamine Barrettet al. (1979) for 2 hours at 37° C. with subsequent dialysis against PBSat 4° C.

Using the above-described thrombolysis assay, it was shown that plasminwas capable of dissolving plasma thrombi in a dose-dependent manner asshown in FIGS. 5 and 6. Increasing amounts of plasmin were added to 1251fibrin-labeled plasma thrombi and the degree of thrombolysis wasassessed by measuring the release of radioactivity: (a) 0.15 mg/ml ofplasmin in the reaction tube, (b) 0.30 mg/ml; (c) 0.45 mg/ml; (d) 0.60mg/ml; (e) control with no plasmin added. The data in FIG. 6 demonstratethat plasmin is also active in a plasma environment and is capable ofdissolving plasma thrombi. This phenomenon is dose-dependent. At thesame time, unlike with the Lys-plasminogen and tPA mixture, thrombolysisby plasmin alone does not progress to completion in a plasmaenvironment; thrombolysis tends to cease after 2 hours. While notwishing to be bound by any one theory, this phenomenon can be explainedby the presence of various protease inhibitors in a plasma environmentand, especially by rapid inhibition of non-thrombus-bound plasmin byα₂-antiplasmin.

To assess the role of plasma inhibitors in plasmin-catalyzed thrombuslysis, a series of thrombolysis experiments was performed with normalplasma and plasma samples lacking α₂-antiplasmin, α₂-macroglobulin orall the inhibitors, or PBS.

As seen from FIG. 6, plasmin-induced fibrinolysis was enhanced bydeletion of either α₂-antiplasmin or α₂-macroglobulin in the plasmasurrounding the thrombus, and even more so by the deletion of allinhibitors. These results confirm that thrombolysis by plasmin is undervery strict physiologic control by the endogenous plasma plasmininhibitors, thus providing a basis for safer thrombolytic therapy.

In contrast to plasminogen activator-induced thrombolysis, thrombolysiswith plasmin can be controlled by these inhibitors. Plasminogenactivator-induced thrombolysis utilizes the internal source ofplasminogen present in plasma that, from the practical point of view, isunlimited in a human body. On the other hand, plasmin-inducedthrombolysis entirely depends on the external source of the plasmin.Cessation of plasmin administration to the patient should result inrapid cessation of thrombolysis and provides a basis for saferthrombolytic therapy.

EXAMPLE 6 Comparison of Reversibly Inactivated Acidified Plasmin and tPAin an in vitro Model of Peripheral Arterial Occlusion (PAO)

Aged, retracted clots are deficient in plasminogen, the substraterequired by all plasminogen activators for effective clot lysis. Robbieet al. Thrombosis and Haemostasis, 1996, 75(1), 127-33. Aged clots aresignificantly less susceptible to lysis by plasminogen activators.

Aged clots differ from clots causing myocardial infarction or strokeboth mechanically and in their protein composition. They are typicallyfound in the peripheral arteries and can grow to a significant size. Theaverage size of a peripheral clot found in the native arteries is14.8±11 cm as described in Ouriel et al. Current thrombolyticsrepresented exclusively by plasminogen activators are almost ineffectivein lysis of these types of clots. Typically, a patient with PAO istreated for more than 24 hours with urokinase and even after thisprolonged time, the complete patency of the vessel is not achieved. Thesituation may be exacerbated when the thrombolytics are delivered viacatheter into the interior of the clot, further preventing the access ofthe plasminogen substrate required for their efficacy.

Another approach to the lysis of retracted clots is the direct deliveryof active plasmin via a catheter, into the interior of the clot.Delivery of reversibly inactivated acidified plasmin directly intoretracted clots circumvents the inherent plasminogen deficiency of theseclots and provides predictable, rapid and effective clot thrombolysisirrespective of plasminogen content. Moreover, the abundant presence ofthe natural plasmin inhibitor, α₂-antiplasmin, in plasma serves toinstantaneously inactivate any plasmin which escapes from the vicinityof the clot, thereby preventing distal fibrinolysis and consequentbleeding episodes.

To compare the efficacy of plasmin and tPA toward the lysis of longretracted clots, we have developed an in vitro model that mimicsparameters of the clots formed in patients with PAO.

In Vitro PAO Model. Fresh whole human blood was collected into 30×0.95cm glass tubes and allowed to clot spontaneously without additives.Tubes were incubated for 20 hours at 37° C. to allow full retraction.Retracted clots were separated from serum using USA Standard testingsieves D16 with 14 mesh and their weights were determined. Blood clotswere transferred into smaller diameter glass tubes that resembled theaverage size clots in leg arteries (0.6×12 cm). A multi-side portpulse-spray catheter (French size 5 with the 11 cm spraying tip, Cook,Inc.) was inserted into the clot and a thrombolytic reversiblyinactivated acidified plasmin composition according to the presentinvention, or tpA) at 1 mg/ml was delivered in 1-ml increments separatedby 1 hour time intervals. The number of injections corresponds to thedose of thrombolytic. The extent of clot lysis was measured by theweight of a residual clot and expressed as a percent of clot weightreduction. This model is called a PAO model. It mimics the dimensions ofthe thrombi found in PAO patients, although venous blood was used forclot formation. Both tPA and the reversibly inactivated acidifiedplasmin composition according to the present invention were tested inthis model and the results are presented below.

Plasmin is as effective as tPA for lysis of fresh clots, unlike when tPAand plasmin are used for lysis of retracted clots aged for 20 hours toallow complete cross-linking by Factor XIII. tPA is unable to lyse suchclots. Clot weight reduction obtained with tPA-treated clots is similarto the control, even when the dose is raised up to 5 mg per clot.

Plasmin, on the other hand, is effective toward fully retracted andcross-linked clots. There is a dose-dependence of the lytic effect ofplasmin and after five injections (or 5 mg plasmin in total) the clotsare almost completely lysed. In a similar series of experiments, thesame inability to dissolve retracted and cross-linked human thrombi wasobserved with urokinase. Locally delivered plasmin therefore is a moreeffective thrombolytic agent than tPA and other plasminogen activators.

These in vitro data show that tPA requires the presence of itssubstrate, plasminogen, in the clot to initiate and maintain clot lysis.Therefore, while plasmin is as effective as tPA for lysing fresh orplasminogen-rich clots, plasmin is more effective that tPA, and otherplasminogen activators, for lysing of long retracted plasminogen-poorclots. Moreover, the data presented in this example demonstrates thatplasmin is effective in its reversibly inactivated acidified form whenit is injected directly inside the clot.

The clots in the glass tubes are also a model wherein a thrombus orfibrin plug forms in catheters placed in a human or animal and thecatheter is occluded.

EXAMPLE 7 Rabbit Jugular Vein Thrombosis Model

The in vivo efficacy and safety of locally administered active plasminwas determined by the rabbit jugular vein thrombosis model of Collen etal., (1983). The model, in brief was as follows: Rabbits (2-3 kg) aresedated with ketamine/xylazine and a 22G venous catheter was placed inthe ear vein for administration of a dilute solution of pentobarbital(5-10 mg/kg/hr). The neck was shaved and an arterial catheter was placedin the carotid artery for pressure measurements and blood sampling. Atracheotomy was performed for placement of a tracheal tube to facilitatebreathing. The jugular vein was carefully isolated with blunt dissectionup to and including the facial vein. A 24G catheter was placed in thefacial vein and the jugular vein clamped both distal and proximal to theisolated facial vein. The isolated vein segment was washed several timeswith saline and completely drained of blood. The isolated segment waswashed several times with a thrombin solution (500 U/ml). Following thelast wash, 0.5 ml of arterial blood sample was drawn from the arterialcatheter and mixed quickly with 50 μl of ¹²⁵I-labelled fibrinogen(approximately 1 μCi). The mixture was rapidly infused into the isolatedvein segment via the facial vein until the vein segment was fullydistended. The thrombus was mixed by massaging the vein with forceps anda piece of Saran Wrap placed over the exposed jugular vein to preventdrying. Heparin (100 IU/kg) was administered to prevent the depositionof cold fibrinogen on thrombus. The thrombus was allowed to age for 30minutes and clips were removed.

The stability of the thrombus is monitored over a 30-minuteequilibration period. Dissolution of the labeled thrombus (% lysis) wasmonitored continuously with a G1LE gamma counter probe placed directlyover the thrombus.

EXAMPLE 8 Restoration of Venous Flow

To provide a physiologic readout of thrombus dissolution, blood flowrestoration was used as another index of thrombolysis in a separateseries of experiments. In these experiments, an appropriately sized flowprobe was placed distal to the occlusive thrombus (not radiolabeled)connected to a Transonic Flowmeter and venous blood flow measurementswere taken every minute.

Baseline blood flow before thrombus formation was 12-18 ml/min and datais represented as % of baseline. The percent restoration of venous bloodflow and the consumption of Factor VIII and fibrinogen are shown at 60minutes in FIG. 7, and at 90 minutes in FIG. 8. At doses of 0.5 mg/kgand 1.0 mg/kg, tPA induced 16±4% and 21±10% restoration of baselineblood flow at 60 minutes, respectively. Plasmin at 1.0 mg/kg and 2.0mg/kg induced 20±1% and 33±1% restoration of baseline blood flow,respectively. At 90 minutes, tpA infusion resulted in 18±3% and 26±11%restoration of blood flow, respectively and plasmin had resulted in25±5% and 34±6% restoration of flow, respectively.

EXAMPLE 9 Cuticle Bleeding Times (CBT)

Cuticle bleeding times at 60 minutes are shown in FIG. 9. Salinetreatment resulted in bleeding times of 13±1 minutes. tPA at doses of0.5 mg/kg and 1.0 mg/kg resulted in bleeding times of 13±2 minutes and25±4 minutes, respectively, whereas plasmin, at doses of 1.0 mg/kg and2.0 mg/kg resulted in bleeding times of 17±3 and 19±1 minutes,respectively. tPA shows a prolonged CBT compared to when reversiblyinactivated acidified plasmin is used, demonstrating that systemicfibrinolysis by tPA activation of endogenous plasminogen is moreextensive than if plasmin is administered and rapidly inhibited by serumfactors.

EXAMPLE 10 Rabbit Fibrinolytic Hemorrhage Model

The greater safety of plasmin, relative to the plasminogen activatortPA, was demonstrated in an established rabbit model of fibrinolyticbleeding that represents a valid indication of fibrinolytic hemorrhage,as described in Marder et al., (1992).

Thrombolytically equivalent doses (10 ml volumes) of plasmin and tpAwere compared in ear-puncture model for their relative effects on theoccurrence and duration of re-bleeding, and on selected plasmacoagulation and fibrinolytic parameters. Two different doses of plasmin(2.0 or 4.0 mg/kg) or tPA (1.0 or 2.0 mg/kg) were infused over 60-minuteperiods into rabbits through a catheter inserted into the externaljugular vein. Ear punctures (approximately six per ear) with a scalpelblade prior to, during, and after infusion of each fibrinolytic agentwere observed for both bleeding time and for re-bleeding. Primarybleeding times were performed at minus 30, minus 10, 0, 10, 30, 60, 70,90, 120 and 180 minutes relative to the start of each infusion. Citratedblood samples (5 ml) were obtained before, during, and for up to twohours after infusion of each fibrinolytic agent; these blood sampleswere assayed for fibrinogen, Factor VIII, and α₂-antiplasmin. Eachexperimental group consisted of five rabbits.

The primary bleeding times tended to be longer for the tPA groups ateach time point after initiation of infusion until the 90-minute point(Table 4).

TABLE 4 Primary bleeding times. Mean Bleeding Time for Group (Minutes)at Different Times (Minutes) Experimental Relative to the Start ofInfusion Group −30 −10 0 10 30 60 70 90 120 180 tPA (1.0 mg/kg) 2.2 1.71.4 3.3 2.8 2.5 1.6 1.6 1.2 2.4 tPA (2.0 mg/kg) 1.7 1.9 1.6 1.6  2.7*3.9  3.0* 2.3 1.7 1.0 Plasmin (2.0 1.6 1.7 1.8 2.0 1.9 2.5 1.8 2.2 2.01.7 mg/kg) Plasmin (4.0 1.5 1.5 1.4 2.3  1.1* 2.8  1.4* 0.9 1.4 1.5mg/kg) *Statistically greater than the corresponding mean valuesobserved with the indicated plasmin groups (p < 0.05).

This effect was statistically significant (p<0.05) in the comparison ofthe high-dose groups of tPA and plasmin at the 30-minute and 70-minuteexperimental times.

Table 5 shows the observed occurrence of re-bleeding in each of the fourexperimental groups.

TABLE 5 Occurrence of Re-bleeding in Each Experimental Group Number ofAnimals Experimental Group Exhibiting Re-Bleeding tPA (1.0 mg/kg) 5 outof 5 tPA (2.0 mg/kg) 4 out of 5 Plasmin (2.0 mg/kg) 0 out of 5 Plasmin(4.0 mg/kg) 0 out of 5 tPA combined groups (n = 10)  9 out of 10 Plasmincombined  0 out of 10

The results distinguish the hemorrhagic activity of the plasminogenactivator, tPA, from that of plasmin. None of the plasmin-treatedanimals exhibited re-bleeding, unlike nine out of ten of the tPA-treatedanimals.

Table 6A summarizes the mean re-bleeding times at active sites and atall sites of re-bleeding for the 1.0 mg/kg tPA dosage group. Table 6Bsummarizes the corresponding data for the 2.0 mg/kg dosage group.

TABLE 6A Mean Re-bleeding Times in Rabbits Receiving tPA at 1.0 mg/kgTime Relative to Start of Infusion (Minutes) −30 −10 0 10 30 60 70 90120 180 Percentage of sites that 60 60 80 100 80 20 20 0 0 0 re-bledMean re-bleeding time 2.0 4.0 31.3 20.7 9.0 1.5 1.0 — — — (minutes) ofactive sites Mean re-bleeding time 1.2 2.4 25.0 20.7 7.2 0.3 0.2 — — —(minutes) of all sites

TABLE 6B Mean Re-bleeding Times in Rabbits Receiving tPA at 2.0 mg/kgTime Relative to Start of Infusion (Minutes) −30 −10 0 10 30 60 70 90120 180 Percentage of sites that 40 80 60 80 60 40 0 0 0 0 re-bled Meanre-bleeding time 32 23 44.7 68.4 48.7 2.5 — — — — (minutes) of activesites Mean re-bleeding time 12.8 18.4 26.8 54.7 7.2 29.2 — — — —(minutes) of all sites

Re-bleeding at active sites showed a distinction between the two dosagegroups for tPA, with longer duration of re-bleeding at the 2.0 mg/kgdose relative to the 1.0 mg/kg dose of tPA. The results from bloodchemistry measurements are summarized in Tables 7-9.

Table 7 presents the levels of fibrinogen measured at the experimentaltimes. The mean fibrinogen levels following tPA infusion fell morerapidly and to a lower nadir than following the plasmin infusions.

TABLE 7 Mean Fibrinogen Levels (mg/dl) in Experimental Groups atDifferent Times (Minutes) Relative to Start of 60-Minute Infusion TimeRelative to Start of Infusion (Minutes) Experimental Group −30 0 10 6070 90 120 180 tPA (1.0 mg/kg) 221 205 202  93 145 126 194 205 tPA (2.0mg/kg) 260 233 151 118 196 190 214 160 Plasmin (2.0 mg/kg) 208 231 214149 167 134 183 145 Plasmin (4.0 mg/kg) 297 293 256 222 146 184 159 140

The mean values for Factor VIII (Table 8) were lower at most of theexperimental times for the tPA-infused animals relative to theplasmin-infused animals, with statistical significance being reached atthe 60-minute experimental time for each tPA group versus each plasmingroup.

TABLE 8 Mean Factor VIII Levels (Percent of Initial) in ExperimentalGroups at Different Times (Minutes) Relative to Start of 60-MinuteInfusion Time Relative to Start of Infusion (Minutes) ExperimentalGroups −30 0 10 60 70 90 120 180 tPA (1.0 mg/kg) (100) 87 84  23* 58 7757 64 tPA (2.0 mg/kg) (100) 85 56  29* 41 36 46 44 Plasmin (2.0 mg/kg)(100) 108 87 77 62 56 72 78 Plasmin (4.0 mg/kg) (100) 85 65 58 58 61 4750 *Statistically lower than the corresponding mean values for either ofthe plasmin groups at 60 minutes (p < 0.05).!

Recovery of Factor VIII in animals receiving tPA was to approximatelythe same concentration (50-60% of initial values) as with animalsreceiving plasmin. Comparison of the two doses of tPA shows a more rapiddecrease in Factor VIII with the higher dose and a more dramatic reboundincrease in Factor VIII with the lower dose.

The mean values for α₂-antiplasmin as shown in Table 9 were lower atmost of the experimental times for the tPA-infused animals relative tothe plasmin-infused animals.

TABLE 9 Mean α₂-Antiplasmin Levels (Percent of Initial) in ExperimentalGroups at Different Times (Minutes) Relative to Start of 60-MinuteInfusion Time Relative to Start of Infusion (Minutes) Experimental Group−30 0 10 60 70 90 120 180 tPA (1.0 mg/kg) (100) 100  39*  4* 34 37 51 43tPA (2.0 mg/kg) (100)  81  12*  0*   9**  15** 15 27 Plasmin (2.0 mg/kg)(100) 104 93 55 55 56 45 79 Plasmin (4.0 mg/kg) (100) 102 89 45 20 49 2424 *Statistically lower than the corresponding mean values for either ofthe plasmin groups at these experimental times (p < 0.05).**Statistically lower than the mean values for the low-dose plasmingroups at these experimental times (p < 0.05).Statistical significance was reached at the 10-minute and 60-minuteexperimental times between each of the tPA groups and either of theplasmin groups and at the 70-minute and 90-minute experimental timesbetween the high-dose tPA groups and the corresponding low-dose plasmingroups.

EXAMPLE 11 Fibrinolysis by Mini-plasmin

Mini-plasmin is a truncated version of plasmin lacking the first fourkringle domains. It can be produced from mini-plasminogen that isgenerated by limited proteolysis of plasminogen with elastase.Mini-plasmin will be more effective for clot lysis than fill-sizedplasmin since: (a) mini-plasmin is smaller than plasmin (38 kDa vs 84kDa) and will more easily diffuse inside the clot; and (b) mini-plasminlacks the α₂-antiplasmin binding site of kringle 1, and therefore willbe resistant to inhibition by α₂-antiplasmin cross-linked to the clot.α₂-Antiplasmin is often responsible for resistance of aged clots tothrombolytic therapy. Mini-plasminogen was purified as described inSottrup-Jensen et al. (1975). Mini-plasminogen conversion intomini-plasmin, its purification and formulation were accomplished usingexactly the protocol as described for plasmin of the Example 2.

The thrombolytic potency of mini-plasmin was compared with that ofplasmin at equimolar concentrations using the in vitro PAO modeldescribed in Example 6. Clots were aged for 20 hours and two 1-ml dosesof plasmin (1 mg/ml) or mini-plasmin (0.45 mg/ml) were delivered to theclot via a catheter. Between injections, clots were incubated at 37° C.for 1 hour. The extent of thrombolysis was measured by clot weightreduction. Clots infused with saline were used as control. Clotdimensions −0.6×12 cm.

As shown in FIG. 10, mini-plasmin causes greater clot lysis than plasminwhen used in the equimolar amounts. The clot weight reduction after two1-ml injections of mini-plasmin at 0.45 mg/ml was 44.3±4.4%. whereas two1-ml injections of plasmin at 1 mg/ml resulted in 36±1.7% clot weightreduction. Mini-plasmin can be an even more effective thrombolytic agentthan plasmin when delivered through the catheter directly to or insidethe clot.

EXAMPLE 12 Efficacy of Localized Catheter Plasmin Administration ofPlasmin

The in vivo efficacy of plasmin (1-2 mg/kg) administered locally via acatheter was compared with that of tPA (0.5 and 1.0 mg/kg) in the rabbitjugular vein thrombosis model. Two approaches were used to assessthrombolysis: 1) real-time measurements of percent lysis with aradiolabeled thrombus; and 2) restoration of baseline blood flow via theapplication of a flow probe and flow meter. The rate of thrombolysis wasmonitored and quantified over 90 minutes, as described in Example 6.Concomitantly, the consumption of Factor VIII and fibrinogen, as well ascuticle bleeding time (CBT), were measured as indicators of the systemiclytic state. For these local administration studies, a catheter (PE 50)was advanced via the marginal ear vein, to within 1 cm of theobstructive thrombus. Two doses of tPA; 0.5 and 1.0 mg/kg and two dosesof plasmin; 1.0 mg/kg and 2 mg/kg were used. These doses were containedin 10.0 ml total volume infused over 30 minutes. The percent lysis at 60and 90 minutes with the radiolabeled thrombus was measured and, inanother separate series of experiments with unlabelled thrombi, percentrestoration of flow was measured at similar time points. Arterial bloodsamples (4 ml) obtained at 0 minutes and 60 minutes were used fordetermination of fibrinogen and Factor VIII levels. Factor VIII levelsand fibrinogen concentrations were determined using a MLA Electra 800Coagulation Timer and the Fibrinogen Assay Set. Factor VIII levels weredetermined by the COATEST VIII C4 assay using human Factor VII togenerate a standard curve. Cuticle bleeding times at 0 minutes and 60minutes were determined by clipping the rabbit's nail, at the apex, witha dog nail trimmer. The blood was dabbed, without touching the nail,with a filter paper every two minutes until a thrombus formed and thefilter paper did not wick blood away. Results are represented asMean±SEM, and to evaluate significant differences between groups aone-way ANOVA followed by Bonferroni's procedure for multiple-comparisontesting was used. P<0.05 was considered significant.

Plasmin or tPA (total volume 10 ml) was infused over 30 minutes via acatheter placed 1 cm proximal to the thrombus. An equal volume of salineserved as a control. Plasmin doses of 1.0-2.0 mg/kg were compared to a0.5 mg/kg dose of tPA. A one-way ANOVA followed by Dunn's method formultiple comparison testing was used for statistical evaluation, (*p<0.05 compared to tPA at 0.5 mg/kg, or # vs tPA at 1.0 mg/kg).

When compared with tPA (0.5 -1.0 mg/kg), plasmin (1.0-2.0 mg/kg) infusedlocally induced comparable or significantly better thrombolysis, withsimilar or less consumption of Factor VIII and fibrinogen and CBT.Risk/benefit evaluation of plasmin treatment revealed that twice thedose of plasmin (on a weight basis) induced similar bleeding sideeffects as did tPA. Thus, plasmin can be effectively and safely used asa thrombolytic agent during catheter-assisted local thrombolysis.Plasmin has comparable or superior lytic activity when compared to tPA,and the safety profile appeared similar in this animal model of localthrombolytic delivery.

The extent of thrombolysis was calculated from the ratio between theamount of radioactivity released from the thrombus into the plasma andthe total amount of radioactivity in the reaction tube. The release oflabeled fibrin degradation products, expressed in percent, was plottedversus time.

The percent lysis of radiolabeled thrombi and consumption of Factor VIIIand fibrinogen are shown at 60 minutes (FIG. 11) and 90 minutesincubation periods (FIG. 12). At 0.5 mg/kg and 1.0 mg/kg, tPA induced16±2% and 21±2% lysis, respectively, whereas plasmin at 1.0 mg/kg and2.0 mg/kg induced 26±3% and 33±4% lysis, respectively. At 90 minutes,tPA infusion had resulted in 21±2% and 26±2% lysis, respectively,compared to plasmin which induced lysis of 31±4% and 36±2%,respectively.

EXAMPLE 13 Risk/benefit Assessment of Plasmin Treatment vs tPATreatment.

An extensive comparative pharmacological evaluation (risk/benefit) ofplasmin in the rabbit model of thrombolysis was determined. The efficacyof plasmin was compared with that of tPA. Percent lysis and restorationof blood flow as indices of efficacy were used. These analysis wereperformed in separate experiments. The respective side effects inducedby these thrombolytic agents were compared by measuring the consumptionof Factor VIII and fibrinogen and assessing bleeding times. This alloweda determination of an approximate risk/benefit profile.

To determine the risk profile, the ED₅₀ dose for tPA was calculated,using the consumption of Factor VIII as a surrogate marker for inductionof the lytic state and bleeding. The consumption of Factor VIII wasdirectly related to the bleeding side effect by replenishing back FactorVIII (Kogenatee) and returning normal hemostasis. tPA doses ranging from0.1 mg/kg to 3.0 mg/kg with a N of at least 10 animals/dose wereexamined. 1.0 mg/kg is considered a clinical dose for tPA. Thecalculated ED₅₀ for tPA was 1.8 mg/kg. A similar calculation for locallyadministered plasmin gave an ED₅₀ of 3.6 mg/kg. Therefore, twice thedose of plasmin (wt basis) induced similar bleeding side effects as tPA.The efficacy (% lysis and blood flow) with doses of tPA and plasmin withequivalent risk profiles i.e., consumption of coagulation proteins andbleeding were evaluated as shown in FIG. 13. At all equivalent riskdoses tested, tPA (0.5-3.0 mg/kg) and plasmin (1.0-3.0 mg/kg), plasmininduced at least similar or significantly better rates of lysis andrestoration of blood flow compared to tPA. The pharmacological data,therefore, is sufficient to recommend that plasmin be considered for thelytic treatment of thrombi accessible by catheter placement.

EXAMPLE 14 Degradation Peptides of Plasmin Samples Characterized byN-terminal Sequencing

The degradation peptides of plasmin samples were characterized byN-terminal sequencing as follows. Plasmin compositions were formulatedat low pH values: a pH less than 2.5 and a pH of 3.5 and 3.8 containing2 mM acetic acid. The plasmin samples were analyzed using SDS-PAGE with4-12% Bis-Tris NuPage gels, as shown in FIG. 14. The protein bands weretransferred to a PVDF membrane, stained with Coomassie Blue R-250(Bio-RAD Laboratories, Hercules, Calif.) and bands cut out using ascalpel.

N-terminal sequence analysis was performed directly from the membraneusing a Hewlett Packard 241 Protein Sequencer (Hewlett Packard, Inc.,Glen Allen, Va.). Ten cycles were run for each band so that thecorresponding fragment of plasmin could be identified. Molecular weightsfor each band were determined with densitometry analysis using the Mark12 marker available from Invitrogen, Inc. (San Diego, Calif.)

Three polypeptides generated by incubation of plasmin at pH 3.8 began atpositions (numbering relative to Lys-plasmin) threonine (T105), glycine(G190) and glutamic acid (E623). From the known amino acid sequence ofplasmin, it was determined that the first two polypeptides were from theheavy chain and the third from the light chain. As shown in FIG. 15, theamino acid preceding the N-terminal amino acid was either arginine orlysine (K104, R189, and K622). It is commonly known that plasmin cleavesproteins on the carboxyl side of lysine and arginine. These resultsdemonstrated that compositions of plasmin at pH 3.8 were susceptible toautodegradation.

Three polypeptides generated by incubation of plasmin at pH 2.2 beganwith proline at the N-termini. From the known amino acid sequence ofplasmin, it was determined that these polypeptides were from the heavychain, starting at positions P63, P155, and P347, as shown in FIG. 15.The amino acid preceding each proline was an aspartic acid (D62, D154,and D346). It is commonly known that aspartyl-prolyl (D-P) peptide bondsare acid labile. These results demonstrated that compositions of plasminat pH 2.2 were susceptible to acid hydrolysis of peptide bonds.

EXAMPLE 15 Sugars in Low pH Formulation Stabilize Plasmin

Plasmin was formulated in one of the following acids with at least oneof the following sugars, at pH 2.5 to 4.0. The group of acids included 1mM to 500 mM of acetic acid, citric acid, serine, threonine, isoleucine,valine, glutamine, P-alanine, or other acids, preferable in the range of1 mM to 50 mM. The group of sugars included 0.2% to 20% of maltose,mannitol, sorbitol, sucrose, lactose, glucose and trehalose. The plasminformulation without any excipient was included as a control. All sampleswere incubated at 37° C. for 7 days. The change in plasmin integrity wasanalyzed by running reducing SDS-PAGE.

The results shown in FIG. 16 demonstrate that sugars have a stabilizingeffect on plasmin at pH 3.5 and improve the benefits from storingreversibly inactivated acidified plasmin at low pH.

EXAMPLE 16 Stabilization of Reversibly Inactivated Acidified PlasminComposition with Non-carbohydrate Stabilizing Agents

Reversibly inactivated acidified compositions were formulated at 1 mg/mlin 5 mM acetic acid, pH 3.7, according to the present invention, with0.1M of glucosamine, niacinamide, citrulline or thiamine added as anon-carbohydrate stabilizer. A reversibly inactivated acidified plasminformulation without any excipient stabilizing agent was included as acontrol. All samples were incubated at 37° C. for 7 days and the changein plasmin integrity analyzed using SDS-PAGE under non-reducingconditions. Referring now to FIG. 17, all of the non-sugar stabilizingagents tested improved the stability of the reversibly inactivatedacidified plasmin composition at 37° C. over the 7day test period.

A reversibly inactivated acidified plasmin compositions was alsoformulated at 1 mg/ml in 2 mM acetic acid, pH 3.4, according to thepresent invention, with 150 mM sodium chloride as a stabilizing agent.The same formulation, but without sodium chloride, was also prepared andincluded as a control. Samples were incubated at 4° C. for 28 days. Thechange in plasmin integrity was analyzed using SDS-PAGE undernon-reducing conditions, as described in Example 3 above. Values werenormalized relative to day 0 controls which were assigned a value of100%. Table 5, the results demonstrate that plasmin stored at 4° C. ismore stable in the low-pH formulation containing sodium chloride.

TABLE 5 Stability of reversibly inactivated acidified plasmincomposition (2 mM NaAc, pH 3.4) with or without 150 mM sodium chloride,stored at 4° C. Sodium chloride % intact % intact Concentration Plasminheavy chain light chain % activity (mM) (mg/ml) after 28 days after 28days after 28 days  0 1  90 93 81 150 1 101 95 97

EXAMPLE 17 Plasma Can Neutralize Proportionally Large Volumes ofAcidified Saline

In order to estimate the volume of reversibly inactivated acidifiedplasmin compositions of the present invention that can be neutralized byone milliliter of serum at physiological pH, a series of titrationexperiments were performed. A 1-ml volume of serum is estimated torepresent the liquid phase volume of an average retracted clot found inthe artery of a PAO patient. Serum (1 ml) was titrated with an acidified(pH 3.7) low buffering capacity buffer solution typical of that in whichreversibly inactivated acidified plasmin according to the presentinvention is formulated. As shown in FIG. 18, 1 ml of serum is capableof neutralizing approximately 150 ml of acidified saline. The lattervolume will exceed the likely volume of reversibly inactivated acidifiedplasmin required for treatment of peripheral arterial occlusions.

Thus, the formulation of reversibly inactivated acidified plasmin in alow buffering capacity buffer provides the basis for an effective andstable pharmaceutical composition. Such compositions would allowadministration of reversibly inactivated acidified plasmin at roomtemperature for the time required for treatment without the plasminsignificantly losing potency during delivery of the preparation to thepatient.

EXAMPLE 18 Trypsin Stabilized at Low pH Can be Reactivated by Transferto Higher pH Environment

Trypsin (16.4 mg, Sigma Chemical Co. Catalog No. T-1426) was dissolvedin 2.28 ml of 50 mM Tris/0.5 M NaCl (pH 8.0). The trypsin solution wasloaded onto a 1-ml column of Benzamidine-Sepharose (Pharmacia Code No.17-0568-01) that had been pre-equilibrated with 50 mM Tris/0.5 M NaCl(pH 8.0). This column was washed with 4 ml of this latter buffer,resulting in a decrease in the eluate absorbance (280 nm) to less than0.03. Trypsin was eluted from this column in an inactivated state with0.5-ml volumes of 200 mM glycine/0.5 M NaCl (pH 3.0); the third throughfifth 0.5-ml fractions eluted from this column contained the peak valuesof absorbance (280 nm) and were pooled. The absorbance (280 nm) of thispooled trypsin eluate was determined to be 9.22; based upon theextinction coefficient for trypsin (E₂₈₀ for a 1% solution=17.09) andthe molecular weight of trypsin (24,000), the concentration of totaltrypsin protein in this pooled column eluate was calculated to be 225μM.

The concentration of trypsin active sites in this pooled trypsin columneluate was determined by the method described by Case & Shaw, Biochem.Biophys. Res. Commun. 29, 508 (1967) and incorporated herein byreference in its entirety, using p-nitrophenylguanidinobenzoate asactive-site titrant. This assay was performed at pH 8.3, by diluting asmall volume (100 μl) of the pooled trypsin column eluate into an assaymixture also containing 700 μl of 50 mM sodium borate (pH 8.3), 200 μlof 10 mM sodium phosphate/1% glycine (pH 7.0) plus 10 μl ofp-nitrophenylguanidininobenzoate (dissolved in dimethyl formamide); thefinal pH of this mixture composition was determined to be 8.3. Thetrypsin-dependent amount of p-nitrophenol formed in this assay wasmonitored at 410 nm. Based upon the extinction coefficient forp-nitrophenol at 410 nm and at pH 8.3 (16,595 M⁻¹), 100 μl of thispooled trypsin column eluate present in the 1.01-ml assay correspondedto a concentration of 22.95 μM trypsin active sites present in thecuvette. Therefore, the original stock solution of pooled trypsin columneluate contained 231 μM trypsin active sites. This latter value isidentical, within experimental error, to the concentration of totaltrypsin protein present (225 μM). These results demonstrate that trypsincan be adjusted to low pH and then transferred to a higher pHenvironment with reactivation of its active site.

REFERENCES

The following references are specifically incorporated herein byreference:

-   Deutsch, D. G. & Mertz, E. T., Science 170, 1095-1096. (1970)-   Robbins, K. C. & Summaria, L. Meth. Enzymol., 19, 257-273 (1970)-   Castellino, F. J. & Powell, JR. Meth. Enzymol., 80, 365-378.(1981)-   Lijnen, H. R., Zamarron, C., Blaber, M, Winkler, M. E. & Collen,    D, J. Biol.Chem. 261, 1253-1258 (1986)-   Wiman, B., Biochem. Journal. 191(1):229-32(1980)-   Barrett, A. J., Brown, M. A., & Sayers, C. A., 181, 401-418 (1979)-   Collen, D, Stassen, J. M. & Verstraete, M., J. Clin. Invest. 71(2),    368-376 (1983)-   Marder, V. J., Shortell, C. K, Fitzpatrick, P. G., Kim, C. &    Oxley, D. Thrombosis Res. 67, 31-40 (1992)

U.S. Pat. Nos: 5,288,489 3,950,513 5,879,923.

1. A method of administering a therapeutic dose of a reversiblyinactivated acidified mammalian serine protease composition to a humanor animal having a thrombotic occlusion, comprising the steps of: a)identifying a human or animal having a thrombotic occlusion; and b)administering parenterally to the human or animal a therapeutic dose ofa pharmaceutically acceptable reversibly inactivate acidified mammalianserine protease composition substantially free o plasminogen activator,wherein the therapeutic dose is delivered into or proximally to thethrombotic occlusion; wherein the mammalian serine protease compositioncomprises a low buffering capacity buffer and has a pH less than about4.0, and wherein the composition is a pharmaceutically acceptablesolution that can be raised to a physiological pH by adding no more thanabout 5 volumes of serum to the solution relative to the volume ofsolution.
 2. The method of claim 1, further comprising the step: c)allowing the administered mammalian serine protease composition tointeract with the thrombotic occlusion.
 3. The method of claim 2,further comprising the steps: d) monitoring the level of vascular flowof the human or animal; and e) repeating steps (a)-(d) until apre-selected level of vascular flow is attained.
 4. The method of claim1, wherein the thrombotic occlusion is a vascular occlusion selectedfrom a coronary thrombosis, deep venous thrombosis, peripheralthrombosis, embolic thrombosis, hepatic vein thrombosis, marasmicthrombosis, sinus thrombosis, venous thrombosis, an arterial thrombosis,an occluded arterio-venal shunt, or an occluded catheter device.
 5. Themethod of claim 1, wherein the reversibly inactivated acidifiedmammalian serine protease composition is lyophilized, and wherein themethod further comprises adding a pharmaceutically acceptable carrier tothe lyophilized mammalian serine protease composition beforeadministration to the human or animal.
 6. The method of claim 1, whereinthe reversibly inactivated acidified mammalian seine proteasecomposition comprises Glu-plasmin, Lys-plasmin, mini-plasmin,micro-plasmin, or a truncated variant thereof.
 7. The method of claim 1,wherein the reversibly inactivated acidified mammalian seine proteasecomposition further comprises an anti-thrombotic agent.
 8. The method ofclaim 1, wherein the low buffering capacity buffer comprises water andat least one pharmaceutically acceptable acid, wherein the acid is anorganic acid selected from a carboxylic acid, an oligopeptide, an aminoacid, or inorganic acid.
 9. The method of claim 8, wherein thepharmaceutically acceptable acid is selected from formic acid, aceticacid, citric acid, glycine, isoleucine, serine, threonine, glutamine,aspartic acid, valine and alanine.
 10. The method of claim 8, whereinthe pharmaceutically acceptable acid is acetic acid or citric acid. 11.The method of claim 8, wherein the pharmaceutically acceptable acid isacetic acid.
 12. The method of claim 1, wherein the reversiblyinactivated acidified mammalian serine protease composition has a pH ofbetween about 2.5 and about 4.0.
 13. The method of claim 1, wherein thereversibly inactivated acidified mammalian serine protease compositionhas a pH of between about 3.0 and about 4.0.
 14. The method of claim 1,wherein the reversibly inactivated acidified mammalian serine proteasecomposition has a pH of between about 3.1 and about 3.5.
 15. The methodof claim 7, wherein the mammalian serine protease is in a concentrationrange of between about 0.01 mg/ml to about 50 mg/ml.
 16. The method ofclaim 7, wherein the mammalian serine protease is in a concentrationrange of between about 0.1 mg/ml to about 10 mg/ml.
 17. The method ofclaim 8, wherein the acid is in a concentration range of between about 1mM to about 500 mM.
 18. The method of claim 8, wherein the acid is in aconcentration range of between about 1 mM and 50 mM.
 19. The method ofclaim 1, wherein the reversibly inactivated acidified mammalian serineprotease composition further comprises a stabilizing agent.
 20. Themethod of claim 19, wherein the stabilizing agent is a pharmaceuticallyacceptable carbohydrate selected from glucose, maltose, mannitol,sorbitol, sucrose, lactose or trehalose.
 21. The method of claim 20,wherein the carbohydrate has a concentration in a range of about 0.2%w/v to about 20% w/v.
 22. The method of claim 20, wherein thecarbohydrate is glucose and the concentration thereof is about 20%. 23.The method of claim 20, wherein the stabilizing agent is selected fromglucosamine, niacinamide and thiamine.
 24. The method of claim 20,wherein the stabilizing agent has a concentration in the range of about0.01M to about 0.1M.
 25. The method of claim 1, wherein the therapeuticdose of the reversibly inactivated acidified mammalian serine proteasecomposition is in a range of between about 0.1 mg plasmin/kg body weightand 10.0 mg plasmin/kg body weight.
 26. The method of claim 1, whereinthe therapeutic dose of the reversibly inactivated acidified mammalianserine protease composition is in a range of between about 0.2 mgplasmin/kg body weight and 4.0 mg plasmin/kg body weight.
 27. The methodof claim 1, wherein the therapeutic dose of the reversibly inactivatedacidified mammalian serine protease composition is in a range of betweenabout 1.0 mg plasmin/kg body weight and 2.0 mg plasmin/kg body weight.28. The method of claim 1, wherein the reversibly inactivated acidifiedmammalian serine protease composition is administered by a catheterdevice.
 29. The method of claim 28, wherein the catheter device isdirected to a vascular occlusion.
 30. The method of claim 29, whereinthe catheter device delivers the reversibly inactivated acidifiedmammalian serine protease composition proximal to, or into, a thromboticocclusion.
 31. The method of claim 29, wherein the catheter deviceenters the thrombotic occlusion and the catheter delivers the reversiblyinactivated acidified mammalian serine protease composition directlyinto the thrombotic occlusion.
 32. The method of claim 1, wherein thesolution can be raised to physiological pH by adding no more than about1 volume of serum to the solution relative to the volume of solution.33. The method of claim 1, wherein the solution can be raised tophysiological pH by adding no more than about 0.7 volumes of serum tothe solution relative to the volume of solution.
 34. The method of claim1, wherein the solution can be raised to physiological pH by adding nomore than about 0.3 volumes of serum to the solution relative to thevolume of solution.
 35. The method of claim 1, wherein the solution canbe raised to physiological pH by adding no more than about 0.1 volumesof serum to the solution relative to the volume of solution.